白念珠菌SSK1突变株对氟康唑敏感性的初步研究

目的探讨氟康唑作用于白念珠菌双组分信号传导途径SSK1突变株SSK21后药物敏感性的变化。方法采用微量液体稀释法和固醇测定法测定野生株(CAF2-1)和突变株(SSK21)的最小抑菌浓度(MIC);并应用RT-PCR观察氟康唑作用前后,SSK1的表达变化。结果氟康唑对CAF2-1的MIC为16μg/mL,对SSK21为0.032μg/mL。加入氟康唑后,CAF2-1的SSK1表达明显增加,60min时达到最多。结论 SSK21对氟康唑高度敏感,SSK1基因及其相关的重要基因与药物敏感性的关系值得进一步研究,从而为新的抗真菌药物和治疗途径的研发提供参考。     

Ssk2 mitogen-activated protein kinase kinase kinase governs divergent patterns of the stress-activated Hog1 signaling pathway in Cryptococcus neoformans

The stress-activated p38/Hog1 mitogen-activated protein kinase (MAPK) pathway is structurally conserved in many diverse organisms, including fungi and mammals, and modulates myriad cellular functions. The Hog1 pathway is uniquely specialized to control differentiation and virulence factors in a majority of clinical Cryptococcus neoformans serotype A and D strains. Here, we identified and characterized the Ssk2 MAPKKK that functions upstream of the MAPKK Pbs2 and the MAPK Hog1 in C. neoformans. The SSK2 gene was identified as a potential component responsible for the difference in Hog1 phosphorylation between the serotype D f1 sibling strains B-3501 and B-3502 through comparative analysis of meiotic maps showing their meiotic segregation patterns of Hog1-dependent sensitivity to the antifungal drug fludioxonil. Ssk2 is the only component of the Hog1 MAPK cascade that is polymorphic between the two strains, and the B-3501 and B-3502 SSK2 alleles were distinguished by two coding sequence changes. Supporting this finding, SSK2 allele exchange completely interchanged the Hog1-controlled signaling patterns, related phenotypes, and virulence levels of strains B-3501 and JEC21. In the serotype A strain H99, disruption of the SSK2 gene enhanced capsule and melanin biosynthesis and mating efficiency, similar to pbs2 and hog1 mutations. Furthermore, ssk2Δ, pbs2Δ, and hog1Δ mutants were hypersensitive to a variety of stresses and resistant to fludioxonil. In agreement with these results, Hog1 phosphorylation was abolished in the ssk2Δ mutant, similar to what occurred in the pbs2Δ mutant. Taken together, these findings indicate that Ssk2 is a critical interface connecting the two-component system and the Pbs2-Hog1 MAPK pathway in C. neoformans.     

Insight into the role of HOG pathway components Ssk2p, Pbs2p, and Hog1p in the opportunistic yeast Candida lusitaniae

In the present study, we have investigated the role of SSK2, PBS2, and HOG1, encoding modules of the high-osmolarity-glycerol mitogen-activated protein kinase pathway in Candida lusitaniae. Functional analysis of mutants indicated that Ssk2p, Pbs2p, and Hog1p are involved in osmotolerance, drug sensitivity, and heavy metal tolerance but not in oxidant stress adaptation.     

Sequential gene disruption in Candida albicans by FLP-mediated site-specific recombination

The genetic manipulation of the human fungal pathogen Candida albicans is difficult because of its diploid genome, the lack of a known sexual phase and its unusual codon usage. We devised a new method for sequential gene disruption in C. albicans that is based on the repeated use of the URA3 marker for selection of transformants and its subsequent deletion by FLP-mediated, site-specific recombination. A cassette was constructed that, in addition to the URA3 selection marker, contained an inducible SAP2P-FLP fusion and was flanked by direct repeats of the minimal FLP recognition site (FRT). This URA3 flipper cassette was used to generate homozygous C. albicans mutants disrupted for both alleles of either the CDR4 gene, encoding an ABC transporter, or the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily. After insertion of the URA3 flipper into the first copy of the target gene, the whole cassette could be efficiently excised by induced FLP-mediated recombination, leaving one FRT site in the disrupted allele of the target gene. The URA3 flipper was then used for another round of mutagenesis to disrupt the second allele. Deletion of the cassette from primary and secondary transformants occurred exclusively by intrachromosomal recombination of the FRT sites flanking the URA3 flipper, whereas interchromosomal recombination between FRT sites on the homologous chromosomes was never observed. This new gene disruption strategy facilitates the generation of specific, homozygous C. albicans mutants as it eliminates the need for a negative selection scheme for marker deletion and minimizes the risk of mitotic recombination in sequential disruption experiments.     

A single MAPKKK regulates the Hog1 MAPK pathway in the pathogenic fungus Candida albicans

The Hog1 mitogen-activated protein kinase (MAPK) plays a central role in stress responses in the human pathogen Candida albicans. Here, we have investigated the MAPK kinase kinase (MAPKKK)-dependent regulation of the pathway. In contrast to the Hog1 pathway in Saccharomyces cerevisiae, which is regulated by three MAPKKKs (Ssk2, Ssk22, and Ste11), our results demonstrate that Hog1 in C. albicans is regulated by a single MAPKKK Ssk2. Deletion of SSK2 results in comparable stress and morphological phenotypes exhibited by hog1Delta cells, and Ssk2 is required for the stress-induced phosphorylation and nuclear accumulation of Hog1, and for Hog1-dependent gene expression. Furthermore, phenotypes associated with deletion of SSK2 can be circumvented by expression of a phosphomimetic mutant of the MAPKK Pbs2, indicating that Ssk2 regulates Hog1 via activation of Pbs2. In S. cerevisiae, the Hog1 pathway is also regulated by the MAPKKK Ste11. However, we can find no connection between Ste11 and the regulation of Hog1 in C. albicans. Furthermore, expression of a chimeric Pbs2 protein containing the Ste11-dependent regulatory region of S. cerevisiae Pbs2, fails to stimulate Ste11-dependent stress signaling in C. albicans. Collectively, our data show that Ssk2 is the sole MAPKKK to relay stress signals to Hog1 in C. albicans and that the MAPK signaling network in C. albicans has diverged significantly from the corresponding network in S. cerevisiae.     

Contributions of the response regulators Ssk1p and Skn7p in the pseudohyphal development, stress adaptation, and drug sensitivity of the opportunistic yeast Candida lusitaniae

We recently characterized the histidine kinase receptor genes of Candida lusitaniae. For the present study, we have further investigated the role of SSK1 and SKN7, encoding response regulators. The results of functional analysis of mutants indicated that Ssk1p is involved in osmotolerance and pseudohyphal development, whereas Skn7p appears crucial for oxidative stress adaptation.     

Tetracycline-regulated gene expression mediated by a novel chimeric repressor that recruits histone deacetylases in mammalian cells

Regulated gene expression will provide important platforms from which gene functions can be investigated and safer means of gene therapy may be developed. Histone deacetylases have recently been shown to play an important role in regulating gene expression. Here we investigated whether a more tightly controlled expression could be achieved by using a novel chimeric repressor that recruits histone deacetylases to a tetracycline-responsive promoter. This chimeric repressor was engineered by fusing the tetracycline repressor (TetR) with an mSin3-interacting domain of human Mad1 and was shown to bind the tetO(2) element with high affinity, and its binding was efficiently abrogated by doxycycline. The chimeric repressor was shown to directly interact with mSin3 of the histone deacetylase complex. This inducible system was further simplified by using a single vector that contained both a chimeric repressor expression cassette and a tetracycline-responsive promoter. When transiently introduced into mammalian cells, the chimeric repressor system exhibited a significantly lower basal level of luciferase activity (up to 25-fold) than that of the TetR control. When stably transfected into HEK 293 cells, the chimeric repressor system was shown to exert a tight control of green fluorescent protein expression in a doxycycline dose- and time-dependent fashion. Therefore, this novel chimeric repressor provides an effective means for more tightly regulated gene expression, and the simplified inducible system may be used for a broad range of basic and clinical studies.     

Ssk1p-Independent Activation of Ssk2p Plays an Important Role in the Osmotic Stress Response in Saccharomyces cerevisiae: Alternative Activation of Ssk2p in Osmotic Stress

In Saccharomyces cerevisiae, external high osmolarity activates the HOG MAPK pathway, which controls various aspects of osmoregulation. MAPKKK Ssk2 is activated by Ssk1 in the SLN1 branch of the osmoregulatory HOG MAPK pathway under hyperosmotic stress. We observed that Ssk2 can be activated independent of Ssk1 upon osmotic shock by an unidentified mechanism. The domain for the Ssk1p-independent activation was identified to be located between the amino acids 177∼240. This region might be involved in the binding of an unknown regulator to Ssk2 which in turn activates Ssk2p without Ssk1p under hyperosmotic stress. The osmotic stress response through the Ssk1p-independent Ssk2p activation is strong, although its duration is short compared with the Ssk1p-dependent activation. The alternative Ssk2p activation is also important for the salt resistance.     

Mechanism of Tet repressor induction by tetracyclines: length compensates for sequence in the alpha8-alpha9 loop

Natural Tet repressor (TetR) variants are alpha-helical proteins bearing a large loop between helices 8 and 9, which is variable in sequence and length. We have deleted this loop consisting of 14 amino acid residues in TetR(D) and rebuilt it stepwise with up to 42 alanine residues. All except the mutant with the longest alanine loop show wild-type repression, but none is inducible with tetracycline. This demonstrates the importance of the alpha8-alpha9 loop and its amino acid sequence for induction. The induction efficiencies increase with loop length, when the more tightly binding inducer anhydrotetracycline is used. The largest increase of inducibility was observed for TetR mutants with loop lengths between eight and 17 alanine residues. Since loop residues Asp/Glu157 and Arg158 are conserved in the natural TetR sequence variants, we constructed a mutant in which all other residues of the loop were replaced by alanine. This mutant exhibits increased anhydrotetracycline induction compared to the corresponding alanine variant. Thus, these residues are important for induction. Binding constants for the anhydrotetracycline-TetR interaction are below the detection level of 10(5) M(-1) for the mutant with a loop of two alanine residues and increase sharply until a loop size of ten residues is reached. TetR variants with longer loops have similar anhydrotetracycline-binding constants, ranging between 2.6 x 10(9) M(-1) and 8.0 x 10(9) M(-1), about 500-fold lower than wild-type TetR. The increase of the affinity occurs at shorter loop lengths than that of inducibility. We conclude that the induction defect of the polyalanine variants arises from two increments: (i) the loop must have a minimal length-to allow efficient inducer binding; (ii) the loop must structurally participate in the conformational change associated with induction.     

Actin recovery and bud emergence in osmotically stressed cells requires the conserved actin interacting mitogen-activated protein kinase kinase kinase Ssk2p/MTK1 and the scaffold protein Spa2p

Osmotic stress causes actin cytoskeleton disassembly, a cell cycle arrest, and activation of the high osmolarity growth mitogen-activated protein kinase pathway. A previous study showed that Ssk2p, a mitogen-activated protein kinase kinase kinase of the high osmolarity growth pathway, promotes actin cytoskeleton recovery to the neck of late cell cycle, osmotically stressed yeast cells. Data presented herein examined the role of Ssk2p in actin recovery early in the cell cycle. We found that actin recovery at all stages of the cell cycle is not controlled by Ssk1p, the known activator of Ssk2p, but required a polarized distribution of Ssk2p as well as its actin-interacting and kinase activity. Stress-induced localization of Ssk2p to the neck required the septin Shs1p, whereas localization to the bud cortex depended on the polarity scaffold protein Spa2p. spa2delta cells, like ssk2delta cells, were defective for actin recovery from osmotic stress. These spa2delta defects could be suppressed by overexpression of catalytically active Ssk2p. Furthermore, Spa2p could be precipitated by GST-Ssk2p from extracts of osmotically stressed cells. The Ssk2p mediated actin recovery pathway seems to be conserved; MTK1, a human mitogen-activated protein kinase kinase kinase of the p38 stress response pathway and Ssk2p homolog, was also able to localize at polarized growth sites, form a complex with actin and Spa2p, and complement actin recovery defects in osmotically stressed ssk2delta and spa2delta yeast cells. We hypothesize that osmotic stress-induced actin disassembly leads to the formation of an Ssk2p-actin complex and the polarized localization of Ssk2p. Polarized Ssk2p associates with the scaffold protein Spa2p in the bud and Shs1p in the neck, allowing Ssk2p to regulate substrates involved in polarized actin assembly.     

The Trichoderma atroviride seb1 (stress response element binding) gene encodes an AGGGG-binding protein which is involved in the response to high osmolarity stress

The chitinase genes of Trichoderma spp. (ech42, chit33, nag1) contain one or more copies of a pentanucleotide element (5'-AGGGG-3') in their 5'-noncoding regions. In Saccharomyces cerevisiae, this motif is recognized and bound by the stress response regulator proteins Msn2p/Msn4p. To test whether this motif in the chitinase promoters is bound by a Trichoderma Msn2/4p homolog, we have cloned a gene (seb1) from T. atroviride which encodes a C2H2 zinc-finger protein that is 62 (64)% identical to S. cerevisiae Msn2p (Msn4p) in the zinc-finger region, and almost identical to the G-box binding protein from Haematonectria haematococca and to polypeptides encoded by uncharacterized ORFs from Neurospora crassa and Aspergillus nidulans. Its zinc-finger domain specifically recognizes the AGGGG sequence of the ech42 and nag1 promoter in band-shift assays. However, a cDNA clone of seb1, when overexpressed in S. cerevisiae, was unable to complement a Delta msn2/4 mutant of S. cerevisiae. Levels of seb1 mRNA increased under conditions of osmotic stress (sorbitol, NaCl) but not under other stress conditions (cadmium sulfate, pH, membrane perturbance). A T. atroviride Delta seb1 strain, produced by transformation with a seb1 copy disrupted by insertion of the A. nidulans amdS gene, showed strongly reduced growth on solid medium, but grew normally in liquid medium. In liquid medium, growth of the disruption strain was significantly more inhibited by the presence of 1 M sorbitol and 1 M NaCl than was that of the wild-type strain. Despite the presence of AGGGG elements in the promoter of the chitinase gene nag1, no differences in its expression were found between the parent and the disruption strain. EMSA analyses with cell-free extracts obtained from the seb1 disruption strain showed the presence of proteins that could bind to the AGGGG-element in nag1 and ech42. We therefore conclude that seb1 encodes a protein that is involved in the osmotic stress response, but not in chitinase gene expression, in T. atroviride.